Biological samples will often contain different types of cells having particular characteristics. Variations in the type of cells or in the characteristics of cells found in biological fluids has been used to diagnose disease and in developing therapeutic protocols. Biological fluids are typically collected and immediately shipped to a laboratory that has the equipment and personnel to process the fluid and determine the presence or absence of particular cell populations in those fluids. A cell population is a collection of cells that may be identified and grouped together because they all express a common cell marker, or molecule that is associated with a type of cell or a characteristic of a cell. However, one of the difficulties in working with living cells is that the cells must be analyzed while they are fresh or they may give spurious results. This can be very difficult when the samples are procured in areas that are difficult or impossible to reach within a short period of time such as certain rural areas, third world countries, and outer space. In fact, biological sample analysis is often foregone because the samples cannot be transported to a laboratory in a timely manner.
One of the most commonly studied biological fluids in blood. The peripheral blood of a normal subject contains red blood cells, also known as erythrocytes, and five major classes of mature white cells. These five classes are known as neutrophils, eosinophils, monocytes, lymphocytes, and basophils. Each type of mature blood cell performs specialized functions necessary in maintaining the homeostasis of the host. The concentration of each class of peripheral blood cells is tightly regulated and monitored by a dynamic process involving a variety of factors present in the microenvironment of the bone marrow.
Under certain disease conditions and therapeutic protocols, the bone marrow may release either an increased or decreased number of certain classes of white cells. Under other conditions and therapeutic protocols, normal regulation of the number of peripheral blood cells released from the bone marrow is perturbed and an uncontrolled number of immature white or red cells are released to the peripheral blood. Therefore, monitoring the concentration of the five normal classes of white cells and identifying the subpopulations of these five normal classes of white cells has become an important diagnostic tool for physicians.
Blood cells, particularly the white cells, exhibit known cell markers that can be used to identify the presence or absence of specific populations, or subpopulations, of white blood cells. For example, the detection of specific cell markers can be used in the diagnosis of particular viral infections. White cells that have been infected with a virus will commonly express some of the coat protein of that virus within its cell membrane. In addition, macrophages that have engulfed infected cells will commonly express those viral coat proteins in their cell membranes. Antibodies to viral antigens, as well as, polynucleic acid probes have been developed and used for detecting cells that have been infected with different types of virus, including for example the Epstein Bar virus and the Hepatitis B virus.
The efficacy of therapeutic regimes and a patient's prognosis may also be assessed by quantifying the number of specific cell populations within the blood, or by quantifying a ratio of specific types of cells. For example, the efficacy of a drug protocol in the treatment of AIDS patients is commonly followed by analyzing their ratio of helper/inducer T lymphocytes (identified by their reaction to a CD4 monoclonal antibody) to suppressor/cytotoxic T lymphocytes (identified by their reaction to a CD8 monoclonal antibody). The response of patients to chemotherapy is also commonly followed by determining the cell differential of patients at various times within their treatment protocol.
A variety of other tests, both diagnostic and predictive, have been developed that take advantage of identifying particular cell markers in cell populations. For example, prenuptial screening for genetic traits is commonly done to assist in the genetic counseling of certain couples who desire to get married and have children. Such genetic screening is commonly performed using DNA probes for known sequences that occur in individuals that have or carry particular genetically inherited traits.
In addition, a number of diagnostic tests that include the identification of specific cell populations expressing particular markers are also used to identify and follow epidemics within a particular animal population. For example, just as viral infections of blood cells may be detected in humans with monoclonal antibodies or other marker identifiers, viral infections of animal populations can also be detected using similar techniques (e.g., the feline leukemia virus).
The determination of cell markers and the use of those markers as a tool for identifying specific blood cell populations has increased as science has expanded its knowledge of cell surface components and the characteristics of subpopulations of lymphocytes, monocytes, neutrophils, eosinophils, and basophils. For example, recent advances in cellular immunology and flow cytometry have been utilized to identify and quantify lymphocyte subclasses such as helper T cells and suppressor T cells. Lymphocyte subclassifications have become an important diagnostic tool, particularly in view of the AIDS epidemic.
Conventional lymphocyte subclassification involves the following steps:
(1) the separation of lymphocytes from other peripheral blood cells by density gradient centrifugation; PA1 (2) the reaction of the lymphocytes with fluorochrome-labelled monoclonal antibodies directed to specific lymphocyte surface antigens; and PA1 (3) the analysis of lymphocyte-antibody reaction products using flow cytometry.
Recently, techniques have become available that bypass the need for density gradient centrifugation to separate the lymphocytes.
Currently, most cell differentiation and lymphocyte immunophenotyping is being done utilizing flow cytometry. The Q-PREP.TM. (manufactured by Coulter Cytometry, Hileah, Fla.) represents an automated methodology for preparing and processing whole blood for flow cytometric analysis. The Q-PREP uses fresh whole blood samples and can be programmed to process multiple samples through a variety of mixing, incubating and washing steps. The Q-PREP is a sophisticated instrument that is impractical to operate in non-laboratory environment.
Alternatively, non-automated processing of whole blood has been done in smaller laboratories or basic research laboratories. These techniques and protocols require the manual pipeting of whole blood into solutions of monoclonal antibodies or other cell marker identifiers. After mixing and incubating these samples, a solution is added to lyse the red blood cells present in the whole blood sample and to fix the reacted white cells. Each step of reagent addition or other manipulation of the blood sample decreases the precision of the overall process and introduces an opportunity for error. Furthermore, the manual procedure is time consuming and requires specialized equipment and technically trained personnel that are generally only available in a laboratory environment.
Immunostaining followed by red cell lysis and white cell fixation is the preferred method for providing flow cytometry samples from fresh whole blood. Current flow cytometric methods require that leukocytes be analyzed free from interference by erythrocytes. In the past, this has been accomplished by density gradient separation of the white cells or by red blood cell lysis with several washing steps. However, it has been reported that centrifugal washing may alter the remaining cellular distribution. The newer lysing solutions, such as FacsLyse.TM. from Becton Dickinson of San Jose, Calif. and Optilyse.TM. C from AMAC (Immunotech) of Westbrook, Me., separate the red blood cell debris and white cells without centrifugation or washing steps. Immunophenotypic analysis of peripheral blood leukocytes and lymphocytes is facilitated by the use of such erythrolytic reagents.
All of the described procedures require collecting and shipping fresh whole blood samples to reference laboratories and clinical centers for processing and analysis within hours of its collection. These centers and laboratories possess the technical expertise and equipment to process and analyze infectious blood samples. Whenever the expedited analysis of fresh blood samples is not possible (i.e., within a few hours of collection), the laboratory analyzing the samples must verify that the holding time and the conditions that the sample has undergone have not destroyed the specimen integrity by comparing the drawn specimen to comparable fresh specimens. Field samples procured off-site in areas that may be difficult or impossible to reach within a short period of time such as certain rural areas, third world countries, or outer space must either be capable of being rushed to a clinical laboratory for analysis, or the sample and the analysis of that sample must be foregone.
In addition, blood is generally collected in glass tubes having a stoppered top. During transport of the fresh blood sample to the laboratory, there is a chance that the collection tube may break or that the top of the tube may loosen allowing the blood to leak out of the tube.